qHTS for induction of synthetic lethality in tumor cells producing 2HG: qHTS for the HT-1080-IDH1KD cell line

Unbiased genomic sequencing for 22 glioma genomes found recurrent mutation of IDH1 on chromosome 2q33, a gene encoding the cytosolic isoform of IIDH1 associated with the tri-carboxylic acid cycle (TCA) that catalyzes the oxidative decarboxylation of isocitrate, yielding alpha-ketoglutarate and CO2 via NADP+ to NADPH conversion. Subsequent studies confirmed the recurrent IDH mutations in up to 70% more ..

Unbiased genomic sequencing for 22 glioma genomes found recurrent mutation of IDH1 on chromosome 2q33, a gene encoding the cytosolic isoform of IIDH1 associated with the tri-carboxylic acid cycle (TCA) that catalyzes the oxidative decarboxylation of isocitrate, yielding alpha-ketoglutarate and CO2 via NADP+ to NADPH conversion. Subsequent studies confirmed the recurrent IDH mutations in up to 70% of secondary gliomas and in 10% of AML cases [1]. In addition, the somatic mutation of cancer-associated IDH1 is a point mutation resulting in various amino-acid substituents at Arginine132 (IDH1 R132), a key residue found in the enzyme's active site that when mutated, results in the loss-of-function in metabolizing isocitrate but confers a gain-of-function to produce the oncometabolite 2-hydroxyglutarate (2HG) [2]. This in effect defines IDH1 as an oncogene and provides an extraordinary opportunity to discover chemical probes against mutant IDH1 that may translate into much needed new therapies for glioma and AML patients.

This assay aims to perform a synthetic-lethal screen for chemical probes specific for 2HG-producing tumor cells using matched paired cell lines (1) HT-1080-NT fibrosarcoma cell line that overproduces 2HG and (2) HT-1080-IDH1KD cell line which has IDH1 expression knocked-down. Desirable compounds are toxic to the NT fibrosarcoma cell line but not to the IDH1KD cell line. The data in this AID corresponds to the results of the qHTS against the HT-1080 IDH1KD cell line.

Suspensions of trypsinized HT-1080-IDH1KD cells were dispensed into white, tissue culture-treated, solid 1536-well plates at 5uL/well (750 cells/well final concentration) in RPMI medium supplemented with 5% FBS. Plates were incubated at 37 degrees C for 2 hours, after which 23 nL of compounds or DMSO were delivered to each well using a pin tool. Plates were then incubated at 37 degrees C for an additional 48 hours. CellTiter-Glo viability reagent (Promega) was dispensed into wells (2.5uL/well) and incubated at room temperature for 10 minutes. The plates were measured on a PerkinElmer ViewLux plate reader for luminescent signal (clear filter, 1 sec exp.). DMSO-treated control wells and media-only (no cells) control wells were used to normalize %Activity of identified toxic compounds; DMSO-treated wells corresponded to 0%Activity (100% viability), while media-only controls were used to normalize 100%Activity (0% viability). To minimize variability between cell lines, both HT-1080-NT and HT-1080-IDH1KD lines were dispensed and treated with identical compound series in single batches.Concentration-response curves were fitted to the signals arising from the resulting luminescence. The concentration-effect curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Active (toxic) compounds showed concentration-dependent decreases in luminescence, concordant with a decrease in ATP levels and overall cell number. Inactive compounds showed no effect on total luminescence.

Compound Ranking:1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.

Float

μM

3

Efficacy

Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.

Float

%

4

Analysis Comment

Annotation/notes on a particular compound's data or its analysis.

String

5

Activity_Score

Activity score.

Integer

6

Curve_Description

A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.

String

7

Fit_LogAC50

The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).

Efficacy at zero concentration of compound from a fit of the data to the Hill equation.

Float

%

12

Fit_CurveClass

Numerical encoding of curve description for the fitted Hill equation.

Float

13

Excluded_Points

Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.